Plants 2015, 4, 63-84; doi:10.3390/plants4010063
OPEN ACCESS
plants
ISSN 2223-7747
www.mdpi.com/journal/plants
Article
Cell Wall Loosening in the Fungus, Phycomyces blakesleeanus
Joseph K. E. Ortega *, Jason T. Truong †, Cindy M. Munoz † and David G. Ramirez †
Bioengineering Laboratory, Mechanical Engineering Department, University of Colorado Denver, Denver,
CO 80217, USA; E-Mails: [email protected] (J.T.T.); [email protected] (C.M.M.);
[email protected] (D.G.R.)
†
These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail: [email protected];
Tel.: +1-303-556-8489; Fax: +1-303-556-2511.
Academic Editor: Philip J. Harris
Received: 24 October 2014 / Accepted: 13 January 2015 / Published: 21 January 2015
Abstract: A considerable amount of research has been conducted to determine how cell
walls are loosened to produce irreversible wall deformation and expansive growth in plant
and algal cells. The same cannot be said about fungal cells. Almost nothing is known about
how fungal cells loosen their walls to produce irreversible wall deformation and expansive
growth. In this study, anoxia is used to chemically isolate the wall from the protoplasm of
the sporangiophores of Phycomyces blakesleeanus. The experimental results provide direct
evidence of the existence of chemistry within the fungal wall that is responsible for wall
loosening, irreversible wall deformation and elongation growth. In addition, constant-tension
extension experiments are conducted on frozen-thawed sporangiophore walls to obtain
insight into the wall chemistry and wall loosening mechanism. It is found that a decrease in
pH to 4.6 produces creep extension in the frozen-thawed sporangiophore wall that is similar,
but not identical, to that found in frozen-thawed higher plant cell walls. Experimental results
from frozen-thawed and boiled sporangiophore walls suggest that protein activity may be
involved in the creep extension.
Keywords: Phycomyces; expansive growth; wall loosening; chemorheology
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1. Introduction
Cellular expansive growth is central to the development, morphogenesis and sensory responses of
plants, algae and fungi. Plant, algal and fungal cells have walls. These cells regulate the mechanical
behavior of their walls during expansive growth to control the growth rate, morphogenesis and growth
responses to environmental stimuli [1–3]. During expansive growth, water uptake produces turgor
pressure that deforms the cell wall. Walls exhibit both irreversible and reversible (elastic) deformation
during expansive growth [4–6]. The wall deformation behavior is similar to that of a Maxwell–Bingham
viscoelastic model, which consists of a dashpot (filled with a Bingham fluid) in series with an elastic
spring [4,6]. The constitutive equation (stress-strain relationship) for a Maxwell–Bingham viscoelastic
model, Equation (1), describes the strain rate as equal to the sum of the irreversible extension rate of the
dashpot filled with a Bingham fluid and the reversible (elastic) extension rate of a spring [4].
1 d𝐿 1
1 d𝜎
= (𝜎 − 𝜎C ) +
𝐿 d𝑡 µ
𝐸 d𝑡
(1)
Strain rate = irreversible extension rate of a dashpot + reversible extension rate of a spring
where L is the length of the viscoelastic model, t is the time, µ is the dynamic viscosity of the Bingham
fluid, σ is the stress, σC is the critical stress of the Bingham fluid and E is the longitudinal elastic modulus
of the spring. The term, (dL/dt)/L, is the strain rate, and the term, (σ − σC)/µ, is the irreversible extension
rate of the dashpot filled with a Bingham fluid, which behaves like a Newtonian fluid after a critical
stress, σC, is exceeded. The term, (dσ/dt)/E, is the reversible (elastic) extension rate of the spring.
Both irreversible and reversible wall deformations are required for expansive growth. Reversible wall
deformation is responsible for most of the turgor pressure production during water uptake. Irreversible
wall deformation is responsible for the permanent increase in cell volume. This wall deformation behavior
is implicit in the augmented growth equation, Equation (2), that was derived from the constitutive equation
for a Maxwell–Bingham model, Equation (1), and which provides a quantitative mathematical description
of the expansive growth rate as a function of the wall deformation rate of cells with walls [4].
1 d𝑉
1 d𝑃
= 𝜙 (𝑃 − 𝑃C ) +
𝑉 d𝑡
 d𝑡
(2)
Volume expansion rate = irreversible wall deformation rate + elastic wall deformation rate
where V is the volume of the cell wall chamber, t is the time, ϕ is the relative wall extensibility, P is the
turgor pressure, PC is the critical turgor pressure and ε is the volumetric elastic modulus. The term,
dV/Vdt, represents the relative rate of change in volume of the cell wall chamber; the term, ϕ (P − PC),
represents the relative irreversible wall deformation rate; and the term, (dP/dt)/ε, represents the relative
reversible (elastic) wall deformation rate.
The augmented growth equation has been adapted and experimentally validated for diffuse growth,
tip growth, intercalary growth, cellular growth within tissue and growth responses to environmental
stimuli [2,5–7]. It is hypothesized that chemistry within the wall breaks and makes load-bearing bonds
between wall polymers to loosen and harden the wall and regulate temporal and spatial irreversible and
elastic wall deformations (chemorheology) during expansive growth and morphogenesis [1,2,5–7].
Furthermore, it is hypothesized that wall chemistry and chemorheological processes control the
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magnitude and behavior of the inclusive biophysical variables within the augmented growth equation to
regulate the expansive growth rate [1,2,5–7]. Interestingly, the augmented growth equation was recently
derived from fundamental thermodynamic principles as a particular case of a more general growth
equation [8], and the evolution of ϕ and ε as a function of dV/Vdt was correctly predicted.
Investigations with higher plant tissue provide experimental evidence indicating acidic pH initiates
wall chemistry that promotes wall loosening [9]. It is shown that low pH produces continuous deformation
(creep) of frozen-thawed cell walls of higher plant cells when a constant stress is applied [10,11]. Thus,
a chemorheological process promotes creep in frozen-thawed cell walls. Investigators have identified
and isolated pH-dependent proteins (expansins) that mediate the wall loosening and creep in frozen-thawed
plant tissue [1,11]. Expansins and their genes have been found in higher plants, fungi and bacteria [1,12].
Importantly, expansins have been shown to promote creep and irreversible wall deformation in frozen-thawed
tissue from many different plants and are hypothesized to play a major role in the expansive growth of
higher plants [1]. Endogenous wall enzymes are also hypothesized to play a role in the expansive growth
of higher plants (e.g., xyloglucan endotransglycosylase/hydrolase and endo-β-1,4-glucanases), but they
have not been shown to promote creep in frozen-thawed cell walls [1,13]. Experimental evidence
indicates that another chemorheological process (calcium pectate cycle) plays a major role in the
expansive growth of pollen tubes [14] and algae [15] and may play a role in the expansive growth of
higher plant cells [3,8].
Importantly, almost nothing is known about the mechanism of wall loosening in fungal cell walls,
although a considerable amount is known about fungal cell wall structure, synthesis and assembly [16–18].
In the current study, experiments are conducted to obtain insight into the wall loosening mechanism of
the large single-celled sporangiophores of the fungus, Phycomyces blakesleeanus [17,18]. The cell wall
mechanical properties of these sporangiophores have been studied extensively during expansive growth
and exhibit wall deformation characteristics that are similar to those of higher plant cell walls [2,6,7]. It
was shown that their walls exhibit deformation behavior similar to that of a Maxwell-Bingham viscoelastic
model and that the augmented growth equations accurately describe their expansive growth rate [2,4,6,7].
In the present experimental investigation, anoxia is used to chemically isolate the wall from the
protoplasm of the sporangiophores of P. blakesleeanus. Anoxia terminates the sporangiophore’s
metabolism [17,19] and essentially stops the export of wall polymers and materials from the protoplasm
to the wall, thus chemically isolating the wall. The experimental results provide direct evidence of the
existence of chemistry and chemorheology within the fungal wall that are responsible for wall loosening,
irreversible wall deformation and elongation growth.
In addition, constant-tension extension experiments are conducted on frozen-thawed sporangiophore
walls to obtain insight into the wall chemistry and chemorheology that produce irreversible wall deformation.
It is found that a decrease in pH to 4.6 produces creep in frozen-thawed sporangiophore walls, similar to
higher plant cells. Frozen-thawed sporangiophore walls that were boiled for 15 s before constant-tension
extension experiments did not exhibit creep, again similar to higher plant cells. This inactivation of
endogenous wall-loosening activity may suggest mediation by an expansin-like protein and/or mechanism.
However, unlike higher plant cells, the creep of the sporangiophores’ walls continues for minutes, not
hours. This result may indicate that the chemistry and chemorheology that produce creep and wall
loosening in the sporangiophore’s wall may be fundamentally different from that in higher plant cells.
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2. Results
2.1. Anoxia Experiments
Growing Stage IV sporangiophores of the fungus P. blakesleeanus (Figure 1) were subjected to
an anoxic environment in order to terminate the sporangiophore’s metabolism [17,19], thus eliminating
the export of wall polymers and materials from the protoplasm to the wall. Experiments were conducted
as follows. A Stage IV sporangiophore was selected and placed in a chamber made of transparent acrylic
that was constructed specifically for these experiments. The sporangiophore was adapted in the
experimental chamber to atmospheric air (~21% oxygen concentration), constant and symmetric
distribution of light and other environmental conditions for 20 min. The light and environmental
conditions were maintained constant throughout the adaptation period and during the experiment to
prevent growth responses. After the adaptation period, the elongation growth was measured for the
remainder of the experiment (Figure 2). Ten minutes afterwards, the sporangiophore was impaled with
the micro-capillary tip of a pressure probe (downward pointing arrow on the time scale and labeled
“Impaled”), and the turgor pressure was measured for the remainder of the experiment. At approximately
27 min on the time scale (shown by the downward point arrow labeled “Anoxia”), the chamber was
filled with nitrogen gas, which decreased the oxygen concentration to less than 1%.
The results presented in Figure 2 show that both the elongation rate (slope of the elongation curve,
ΔL) and turgor pressure (P) remain nearly constant between 10 min and 27 min on the time scale. The
sharp and immediate decrease in turgor pressure occurs at the exact time when the oxygen concentration
decreased from 21% to less than 1%. It can be seen that the turgor pressure continues to decrease for
the remainder of the experiment. Interestingly, the turgor pressure decreases slowly for approximately
40 min and then suddenly decreases exponentially. This turgor pressure behavior was typical of more
than five experiments conducted.
The elongation decreases to zero within a few minutes after the initiation of anoxia and continues to
decrease slowly for another 40–50 min. Afterwards, the elongation decreases faster for the remainder
of the experiment until the sporangium falls over onto the stalk. The decrease in elongation represents a
decrease in the length of the sporangiophore stalk. The shrinking in length can be explained as
“recovered” elastic wall deformation that occurs when the turgor pressure decreases in magnitude. This
recovered elastic wall deformation will obscure any irreversible wall deformation (expansive growth)
that may occur during anoxia. It is apparent that the turgor pressure must remain constant in order to
measure irreversible wall deformation.
The pressure probe was used to measure and control the turgor pressure in the sporangiophore
before and during anoxia. Figure 3 shows the results from one of these experiments. As before, a
Stage IV sporangiophore was adapted to constant environmental conditions in the chamber for 20 min
before elongation measurements began, and the conditions remained constant for the remainder of the
experiment. The turgor pressure measurements began at approximately 12 min on the time scale, and
the oxygen concentration was reduced to less than 1% at 25 min on the time scale. The turgor pressure
was held constant at the value that was measured before the initiation of anoxia. Careful inspection of
the elongation curve shows that elongation continues slowly for 15 min after the initiation of anoxia.
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Figure 1. The schematic illustration (left) and corresponding photograph (right) show the
developmental stages of the sporangiophore of P. blakesleeanus. Sporangiophore development
is divided into five stages (Stages I, II, III, IV and V), and Stage IV is further divided into three
sub-stages (IVa, IVb and IVc). The sporangiophores exhibit expansive growth and irreversible
wall deformation in a region termed the “growth zone”. The growth zone is located at the apical tip
(tip growth) in Stage I and Stage II and adjacent to the sporangium (intercalary growth) in
Stage IV. The Stage I sporangiophore is a single pointed cell that grows longitudinally at the apical
tip in the growth zone, 1–2 mm in length. In the schematic, the growth zone is shown as light green
and the non-growing stalk is dark green. Clockwise rotation (when viewed from above) and
elongation growth occur concurrently during Stage I, producing left-handed helical growth.
Stage II begins with spherical growth at the apical tip without elongation and rotation growth.
The diameter of the sporangium continues to increase until Stage III, where the diameter is constant
(~0.5 mm). During Stage III, there is no visible expansive growth. Stage IVa begins with elongation
growth concurrent with counter-clockwise rotation growth (right-handed helical growth) in a short
growth zone located approximately 0.6 mm below the sporangium. The growth zone, elongation
growth rate and rotation growth rate increase in magnitude as the right-handed helical growth
continues for approximately one hour. Then, the rotation rate gradually decreases to zero, and
clockwise rotation begins without interruption of elongation growth. Stage IVb begins with the
initiation of left-handed helical growth. Stage IVb exhibits nearly constant growth zone length
(~2.5 mm), elongation growth rates (~35 μm·min−1) and rotation growth rates (~12 degrees·min−1) for
many hours. Stage IVb sporangiophores are typically used for most biophysical experimental studies.
Stage IVc is initiated by counter-clockwise rotation and right-handed helical growth. Stage V
does not exhibit visible expansive growth. The large cylindrical single-celled sporangiophores are
approximately 150 μm in diameter and can grow in length to ten or more centimeters. The sporangiophores
can detect many environmental stimuli (e.g., gravitational acceleration, ethylene, mechanical stretch,
gases, temperature, wind, light intensity, spatially asymmetric distribution of light, the presence of
solid objects and turgor pressure) and respond to these stimuli with symmetric and asymmetric
changes in expansive growth rate (e.g., growth responses and tropic responses) [7,17,18].
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Figure 2. The change in elongation, ΔL, and turgor pressure, P, of a single Stage IV
sporangiophore is plotted against the same time scale. Turgor pressure measurements begin
at 10 min on the time scale and are marked by the downward pointing arrow labeled
“Impaled”. The turgor pressure curve (gray) is the trace from the strip-chart recorder that
measures the output of the pressure transducer in the pressure probe. The oxygen concentration
is decreased from 21% to less than 1% at approximately 27 min on the time scale and is
marked by the downward pointing arrow labeled “Anoxia”. The immediate small decrease
in turgor pressure from a constant value indicates the exact time when the oxygen concentration
was decreased to less than 1%. Jessica E.C. Olson and Elena L. Ortega (two graduate students)
conducted this experiment while working in Joseph K.E. Ortega’s laboratory.
Figure 3. Both the change in elongation and turgor pressure of a Stage IV sporangiophore
are plotted as a function of time. The turgor pressure was held constant with the pressure
probe during anoxia.
68
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There was some concern that the oil-cell sap interface within the micro-capillary tip of the pressure
probe was unable to move while the turgor pressure was held constant during anoxia. The cell sap of the
sporangiophore is very sticky and has a tendency to adhere to the inner wall of the micro-capillary tip
and prevents an accurate measurement of the turgor pressure. In order to prevent the oil-cell sap from
sticking in one location, small step-ups in turgor pressure were produced by injecting inert silicon oil
inside the micro-capillary tip into the vacuole of the sporangiophore (Figure 4). As can be seen, turgor
pressure measurements began at approximately ten minutes on the time scale, and an anoxic environment
was produced at 22 min. Careful inspection of the elongation curve reveals that elongation growth
occurred 2–5 min after each turgor pressure step-up (when P = constant) until approximately 45 min.
The elongation that occurs one minute after the step-up in turgor pressure is predominately an elastic
response and is not used to determine elongation growth and irreversible wall deformation. For this
experiment, it is concluded that elongation and irreversible wall deformation continue for 23 min
after anoxia.
Similar step-up experiments were conducted on Stage I sporangiophores (Figure 5). The Stage I
sporangiophore was impaled to measure the turgor pressure at 10 min on the time scale, and anoxia
was produced at 21 min. Inspection of the elongation curve shows that elongation growth and irreversible
wall extensibility continued until approximately 27 min, or 6 min after anoxia. It was found that the
duration of elongation growth and irreversible wall deformation after anoxia was shorter for Stage I
sporangiophores compared to Stage IV sporangiophores.
Figure 4. Both the change in elongation and turgor pressure of a Stage IV sporangiophore
are plotted as a function of time. Approximately 5 min after anoxia, turgor pressure step-ups
(approximately 0.021 MPa in magnitude and 5 min in duration) were produced with the
pressure probe.
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Figure 5. Change in elongation and turgor pressure for a Stage I sporangiophore are plotted
as a function of time. Two minutes after anoxia, turgor pressure step-ups (approximately
0.021 MPa in magnitude and 2 min in duration) were produced with the pressure probe.
Some of the results of these experiments are summarized in Table 1, which compares the “duration
of elongation during anoxia” obtained from Stage IV sporangiophores when the turgor pressure is held
constant and when the turgor pressure is stepped up. Results of Student’s t-test indicate that there is not
a significant difference between the values obtained when P was held constant and when P was stepped-up.
Because the values are statistically the same, they are combined. The results indicate that, on average,
the Stage IV sporangiophore continues to exhibit elongation growth and irreversible wall deformation
for approximately 17 min during anoxia. Because the Student’s t-test indicates that there is no significant
difference between the durations obtained from experiments when P was constant and when P was
stepped up, only the step-up in P experiments was conducted for Stage I sporangiophores. The results
presented in Table 1 indicate that the duration of elongation growth for Stage I sporangiophores is smaller
than the corresponding values obtained from Stage IV sporangiophores. Student’s t-tests confirm this
observation: the duration of elongation during anoxia of Stage I sporangiophores is significantly smaller
(p < 0.05) than that obtained from Stage IV sporangiophores when P was constant, when P was stepped
up and when the values were combined.
Table 1. Duration of elongation growth during anoxia for Stage IV and Stage I sporangiophores.
Stage
IV
IV
IV
I
Turgor Pressure during Anoxia
P = constant
Step-up in P
Combined
Step-up in P
Elongation Duration during Anoxia (min)
14.8 ±2.1 SE (n = 5)
18.7 ±3.7 SE (n = 6)
16.9 ±2.1 SE (n = 11)
10.0 ±1.2 SE (n = 9)
2.2. Frozen-Thawed Sporangiophores
Constant-tension extension experiments were conducted on frozen and then thawed walls of the Stage
IV sporangiophore to obtain insight into the wall chemistry and chemorheological process. A five
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millimeter-long section of the wall from a Stage IV sporangiophore, which includes the growth zone,
was adapted in an experimental chamber to a bathing solution of pure water (70 mL, pH = 7, at 20 °C)
for 20 min. Then, a tensile load of 1.24 g was applied to the wall. The tensile load produces longitudinal
stress and longitudinal extension of the wall. After the extension, the length remained constant for four
minutes without creep; see Figure 6 (0–4 min). At four minutes on the time scale, the pH of the solution
was lowered to 4.6 by adding a predetermined amount of pH Red 4.0 buffer to the bathing water.
Immediately, the wall extends (red curve with X data points and labeled “Frozen-thawed”) and continues
to extend for six minutes (4–10 min), exhibiting five minutes of creep (5–10 min). Also shown in Figure 6
are the results of another experiment where the same experimental protocol was conducted on another
frozen-thawed wall, except that the frozen-thawed wall was immersed in boiling water for 15 s before being
subjected to the experimental protocol (blue curve with • data points and labeled “Frozen-thawed-boiled”).
It can be seen that the wall extends after the pH of the bathing solution was reduced to 4.6; however, the
initial extension is small, and the wall does not continue to extend afterwards, i.e., it does not creep.
These experiments were repeated many times. Figure 7 is a plot of the average change in extension
at each minute on the time scale of 15 different frozen-thawed walls (red curve with X data points and
labeled “Frozen-thawed”) and eight frozen-thawed-boiled walls (blue curve with • data points and
labeled “Frozen-thawed-boiled”). The results plotted in Figure 7 indicate that the frozen-thawed walls
creep for eight minutes (5–13 min) after the pH of the bathing solution is reduced from 7.0 to 4.6.
Figure 6. Extension behavior of two 5 mm-long sections from two different Stage IV
sporangiophores that were frozen and then thawed is shown. Each wall section includes the
growth zone of a Stage IV sporangiophore. The red curve with X data points and labeled
“Frozen-thawed” shows the extension behavior of a frozen-thawed wall before and after the
pH of the bathing solution was reduced from 7.0 to 4.6. The blue curve with • data points
and labeled “Frozen-thawed-boiled” shows the extension behavior of a frozen-thawed wall
that was boiled in water for 15 s before being subjected to the same experimental protocol.
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Figure 7. The average change in extension versus time of 15 different frozen-thawed walls
is shown (red curve with X data points and labeled “Frozen-thawed”). The average change
in extension versus time of eight frozen-thawed-boiled walls (blue curve with • data points
and labeled “Frozen-thawed-boiled”) is shown.
Student’s t-tests were conducted to determine whether the extension rates obtained from frozen-thawed
walls after the pH is lowered to 4.6 are significantly different from those of frozen-thawed-boiled walls.
The results are shown in Table 2. The results indicate that the extension rates for the frozen-thawed walls
are significantly higher compared to the frozen-thawed-boiled walls (p < 0.05) for 2–5 min after the pH
was reduced. The interpretation is that creep occurs for four minutes (2–5 min) after the pH was reduced
to 4.6. Fewer results indicate that in some experiments, there is creep until the ninth minute after the pH
is lowered to 4.6.
Table 2. Extension rate for each minute and the result of the Student’s t-tests for frozen-thawed
and frozen-thawed-boiled cell walls.
Minutes after
pH = 4.6
1
2
3
4
5
6
7
8
9
Frozen-Thawed Extension
Rate (μm/min)
191 ±109.1 SE (n = 13)
74.6 ±32.7 SE (n = 9)
17.6 ±5.4 SE (n = 7)
6.0 ±2.8 SE (n = 3)
59.6 ±27.1 SE (n = 7)
43.0 ±45.9 SE (n = 3)
10.0 (n = 1)
5.0 (n = 2)
181.0 (n = 1)
Frozen-Thawed-Boiled
Extension Rate (μm/min)
23.3 ±2.8 SE (n = 8)
0
0
0
0
0
0
0
0
Student’s
t-Test
p = 0.77
p = 0.024
p = 0.005
p = 0.018
p = 0.024
p = 0.154
----------------
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3. Discussion
3.1. Anoxia Experiments
Anoxia was used to chemically isolate the wall from the protoplasm of the sporangiophores of
P. blakesleeanus and to reveal the wall chemistry and chemorheology that produces irreversible wall
extension and elongation growth. The sporangiophores of P. blakesleeanus strictly require oxygen for
growth and development [17,19]. The relationship between elongation growth and the metabolism of the
sporangiophores was studied by using anoxia to inhibit oxidative phosphorylation [17]. Bergman et al. [17]
report, “Many fungi can grow anaerobically for long periods of time, but Phycomyces sporangiophores
do not have this capacity. When oxygen is removed from sporangiophores by placing them in nitrogen,
streaming and growth rapidly stop.” Studies conducted in our laboratory by Morgan A. Scott (Master’s
report, see Acknowledgments) show that streaming inside the sporangiophore stops within a minute;
t = 39.5 ±2.4 (SE) s, n = 10. The finding that the turgor pressure continually decreases after the initiation
of anoxia is consistent with the results of earlier studies that anoxia terminates the protoplast’s metabolism.
The finding that elongation growth continues during anoxia for ten minutes or longer when the turgor
pressure is held constant with the use of a pressure probe demonstrates that a wall chemistry continues
the chemorheological process to produce irreversible wall deformation during anoxia. The wall deformation
is irreversible, because the elongation growth occurs when the turgor pressure is constant (Figures 3–5).
Inspection of the previously-established augmented growth equation, Equation (2), can show this. The
augmented growth equation can be modified to describe the elongation growth of cells with a growth
zone, such as the sporangiophores of P. blakesleeanus; Equation (3) [20].
𝐿g d𝑃 𝐿s d𝑃
d𝐿
= 𝑚g (𝑃 − 𝑃C ) +
+
d𝑡
𝐸g d𝑡
𝐸s d𝑡
(3)
Elongation rate = irreversible rate in growth zone + elastic rate in growth zone +
elastic rate in stalk
where L is the length of the sporangiophore, dL/dt is the elongation rate, mg is the longitudinal
irreversible wall extensibility of the growth zone, P is the turgor pressure, PC is the critical turgor
pressure and Lg and Ls are the lengths of the growth zone and stalk, respectively. Eg and Es are the
longitudinal components of the volumetric elastic modulus within the growth zone and non-growing
stalk, respectively. The term, mg (P − PC), represents the longitudinal irreversible deformation rate of
the wall in the growth zone; the term, (Lg/Eg) dP/dt, represents the longitudinal elastic deformation rate
of the wall in the growth zone; and the term, (Ls/Es) dP/dt, represents the longitudinal elastic deformation
rate of the wall in the non-growing stalk.
It is noted in Equation (3) that when the turgor pressure is constant (P = constant, then dP/dt = 0),
the elongation rate is only a function of the irreversible longitudinal deformation rate of the wall in
the growth zone; Equation (4). Equation 4 is essentially the same equation previously derived by
Lockhart [21]. Thus, the elongation rate that occurs when P = constant (for 15 min after the initiation of
anoxia in Figure 3, between 2–5 min after the first five turgor pressure step-ups in Figure 4 and after the
first two turgor pressure step-ups in Figure 5) represents the irreversible wall deformation rate.
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d𝐿
= 𝑚g (𝑃 − 𝑃C )
d𝑡
(4)
Elongation rate = irreversible longitudinal deformation rate in growth zone
The irreversible wall deformation and elongation growth that occur during anoxia are direct evidence
of a wall-loosening chemistry or chemorheological process. In the sporangiophores, the wall chemistry
and chemorheological process continue for a limited duration during anoxia, typically 10–20 min. Two
explanations for the limited duration of elongation growth and associated wall-loosening chemistry
during anoxia are considered. The first explanation is that irreversible wall deformation continues during
the wall hardening process that begins at the initiation of anoxia. The wall hardening process converts
an irreversible extensible wall to a reversible extensible wall by making bonds between microfibrils and
other wall polymers. It is predicted that the irreversible wall deformation stops when the wall hardening
process is complete. Second, wall building polymers and other relevant wall materials extruded into the
periplasm and inner wall surface before the initiation of anoxia, are used to continue the wall-loosening
chemistry and maintain the irreversible wall extensibility. After these polymers and materials are depleted,
then the wall hardening process begins and continues until completion.
The first explanation predicts that the duration of elongation growth during anoxia for Stage I and
Stage IV sporangiophores are approximately the same, because it is expected that the wall hardening
process for both stages is similar and the rate of hardening is approximately the same. The second
explanation predicts that the duration of elongation growth for Stage IV sporangiophores is larger than
those of Stage I sporangiophores, because the elongation growth rates of Stage IV sporangiophores
(34.1 ± 3.1 (SE) μm·min−1, n = 20) are significantly larger than those of Stage I sporangiophores
(7.1 ± 0.7 (SE) μm·min−1, n = 17) [7]. The larger elongation growth rates of Stage IV sporangiophores
require a faster delivery rate of wall-building polymers and relevant wall-building materials to the periplasm
and inner wall surface compared to Stage I sporangiophores. It is reasonable to expect that the amount
of wall-building polymers and relevant wall-building materials within the periplasm and inner wall
surface at the time anoxia is initiated (and the wall is chemically isolated) is larger for Stage IV
sporangiophores compared to Stage I sporangiophores. If the amount of wall building substrates for the
wall chemistry is larger for Stage IV, it is predicted that the duration of the wall chemistry, wall loosening,
irreversible deformation and elongation growth will be longer for Stage IV compared to Stage I. The
second prediction is consistent with the results presented in Table 1.
3.2. Frozen-Thawed Sporangiophores
Extension experiments were conducted on frozen-thawed sporangiophore walls to obtain insight into
the wall loosening chemistry. The constitutive equation for a Maxwell–Bingham viscoelastic model,
Equation (1), can be modified to assist in the interpretation of the results. Equation (5) is derived from
Equation (1) and describes the longitudinal deformation (extension) rate of the frozen-thawed wall section
after a tensile load, or longitudinal stress (σ), is applied.
d𝐿
𝐿o d𝜎
= 𝑚f (𝜎 − 𝜎C ) +
d𝑡
𝐸f d𝑡
Longitudinal deformation rate = irreversible deformation rate + elastic deformation rate
(5)
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where L is the length of the frozen-thawed wall section, dL/dt is the longitudinal deformation (extension)
rate, mf is the longitudinal irreversible wall extensibility of the frozen-thawed growth zone wall section,
σ is the longitudinal stress, σC is the critical longitudinal stress, Lo is the initial length and Ef is the
longitudinal elastic modulus. The term, mf (σ – σC), represents the longitudinal irreversible deformation
rate of the wall in the growth zone, and the term, (Lo/Ef) dσ/dt, represents the longitudinal elastic
deformation rate of the wall in the growth zone.
Equation (5) is similar to an equation previously used by Takahashi et al. [22] to analyze the
relationship between the extension behavior of frozen-thawed tissue sections of cucumber hypocotyls
and expansin. Here, in Equation (5), we explicitly recognize that mf (longitudinal irreversible wall
extensibility that results from unidirectional stress) is not equal to ϕ or mg (irreversible wall extensibility
or the longitudinal component of the irreversible wall extensibility, respectively, that results from
multidirectional stresses produced by turgor pressure in vivo), and Ef (the longitudinal elastic modulus
that results from unidirectional stress) is not equal to ε (the volumetric elastic modulus that results from
multidirectional stresses produced by turgor pressure in vivo).
Constant tension-extension experiments were conducted using a tensile force of 1.24 gf, or 0.012 N
(F = mass × gravitational acceleration = (1.24 × 10−3 kg) (9.81 m·s−2) = 0.012 N) that was applied to
the frozen-thawed walls. The applied longitudinal wall stress can be estimated using an average
sporangiophore diameter of D = 150 µm and a wall thickness of τ = 0.6 µm; σ = F/πDτ  42 MPa.
Immediately after applying the longitudinal wall stress (tensile force of 0.012 N), initial longitudinal
wall deformations (extensions) ranged from 1–2 mm. The initial wall extension in response to the
application of the wall stress can be determined using Equation (5), where dσ/dt is finite and relatively
large (typically, the wall stress of 42 MPa is applied within a few seconds). It is apparent that the initial
longitudinal extensions are predominately elastic; dL/dt = (Lo/Ef) dσ/dt. After the initial longitudinal
extension, the longitudinal stress and length of the frozen-thawed wall section remained constant.
Because σ is constant in Equation (5) (i.e., dσ/dt = 0), subsequent extensions that occur after decreasing
the pH of the bathing solution to 4.6 represent irreversible deformations and creep, Equation (6).
d𝐿
= 𝑚f (𝜎 − 𝜎C )
d𝑡
(6)
Longitudinal deformation rate = Irreversible deformation rate
The applied longitudinal wall stress, σ  42 MPa, is larger than the estimated longitudinal
stress produced by the turgor pressure in vivo. Previously, the pressure probe was used to determine
the average turgor pressure (P = 0.32 ± 0.01 (SE) MPa, n = 20) and average critical turgor pressure
(PC = 0.26 ± 0.01 (SE) MPa, n = 20) ([7] and the references within). The longitudinal stress produced
by turgor pressure can be estimated using the equation, σ = PR/2τ, where P is the turgor pressure, R is
the radius (75 µm) and τ is the wall thickness (0.6 µm). The longitudinal wall stress produced by the
average turgor pressure (P = 0.32 MPa) is estimated to be, σ = 20 MPa, which exceeds the estimated
critical wall stress produced by the average critical turgor pressure (PC = 0.26 MPa), σC  16.3 MPa.
The objective of the constant-tension extension experiments conducted on frozen-thawed sporangiophore
walls is to learn if lowering the pH of the bathing solution will elicit irreversible extension and creep,
similar to what was found in higher plant cells. The results shown in Figures 6 and 7 (red curve with X
data points and labeled “Frozen-thawed”) demonstrate that lowering the pH of the bathing solution from
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7.0 to 4.6 elicits a chemorheological process, irreversible wall extension (see Equation (6)) and wall
creep. In contrast, if the wall sections were immersed in boiling water for 15 s before the constant
tension-extension experiments, they did exhibit a nearly instantaneous irreversible deformation, but did
not exhibit creep, after the pH of the bathing solution was reduced to 4.6.
Constant-tension extension experiments were previously conducted on frozen-thawed sections of growing
cucumber (Cucumis sativus L.) hypocotyls and oat (Avena sativa L.) coleoptiles immersed in a bathing
solution [10,11]. When the pH of the bathing solution was reduced to 4.5, the frozen-thawed walls begin
to extend, and the extension continued for hours afterwards, thus exhibiting creep. The experimental
results are consistent with the “acid growth” hypothesis [9], which states that plant cells excrete protons
onto the inner surface of the cell wall to decrease the pH and activate an “unknown” wall loosening
process. When the frozen-thawed cucumber and oat walls were boiled in water for 15 s before the
extension experiment, the walls did not exhibit creep when the pH of the bathing solution was lowered
to 4.5. These and other experimental results suggested that protein activity may mediate wall loosening
at low pH. Subsequently, wall-loosening proteins were isolated that are not enzymes, expansins [1].
Expansins are hypothesized to catalyze wall stress relaxation and irreversible wall deformation directly
by disrupting hydrogen bonds between the cellulose microfibrils and hemicellulose [23]. Expansins
have been found in a variety of plants and in bacteria and fungi [1,12]. Crude extracts and purified
expansins have been shown to mediate “acid-induced” extension of boiled isolated primary walls of a
variety of plants [1]. Wall loosening by expansins is consistent with the acid growth hypothesis.
Expansins are thought to be strong candidates for primary wall-loosening agents for cells in the organs
of higher plants (e.g., stems, roots and leaves). However, endogenous wall enzymes, such as xyloglucan
endotransglycosylase/hydrolase and endo-β-1,4-glucanases, are also hypothesized to play an important
role in expansive growth of higher plants [13]. Importantly, a considerable amount of experimental
evidence indicates that pectin plays a major role in the expansive growth of pollen tubes ([14] and the
references within) and algae ([15] and the references within). Furthermore, pectin may play a role in the
expansive growth of higher plant cells [3,8]. Interestingly, a small amount of pectin is found in the
sporangiophore’s cell wall [24], and one wonders if pectin plays a role in the expansive growth of
sporangiophores and fungal cells in general.
The general structure of the sporangiophore’s wall can be described as chitin and β-glucans
microfibrils embedded in an amorphous matrix composed predominately of chitosan, glycoproteins and
lipids [16,18], with a small amount of pectin [24]. The wall polymers are linked together by covalent
bonds, hydrogen bonds, hydrophobic interaction and ionic associations [16]. The microfibrils are extruded
onto the inner wall surface by chitin synthases embedded in the plasma membrane [16]. Chitin synthases
and other enzymes and wall polymers are transported to the plasma membrane in vesicles (chitosomes)
and delivered to the periplasm via exocytosis [16]. Importantly, fibrillogenesis and microfibril networks
have been shown to occur in vitro by incubating purified chitin synthases with substrate (UDP-GLcNAc)
and activators [25,26]. Furthermore, more recently, the genome of P. blakesleeanus was sequenced, and
genes involved in sensory growth responses and cell wall components are being characterized
(http://genome.jgi-psf.org/Phybl2/Phybl2.home.html).
Generally, the microfibrils can be cross-linked directly (by hydrogen bonds, hydrophobic interaction
and ionic associations) and by matrix polymers (chitosan, glycoproteins, and pectin). We hypothesize
that wall chemistry and molecular agents that effectively break and make load-bearing cross-links between
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microfibrils produce chemorheology that regulates wall mechanical properties and wall deformation
behavior. Breaking the cross-links directly, or by disconnecting the microfibrils from the matrix network
and/or each other, will allow the microfibrils to separate and/or slide passed each other when the wall is
stressed by turgor pressure in vivo and by an applied stress in extension experiments. In turn, this
chemorheological process can produce a controlled polymer creep, often referred to as “wall loosening”.
Load-bearing cross-links that are not broken or that were broken and reconnected (after slippage and/or
separation of microfibrils) and subsequently become load-bearing produce the elastic behavior of the
wall. The wall loosening will initiate wall stress relaxation and turgor pressure relaxation that, in turn,
produces water uptake, irreversible wall deformation and an increase in cell volume.
The extension curves obtained from frozen-thawed and frozen-thawed-boiled sporangiophore walls
after the pH is lowered to 4.6 are qualitatively similar to those obtained for cucumber (Cucumis sativus L.)
hypocotyls and oat (Avena sativa L.) coleoptiles [10,11]. The experimental results from the frozen-thawed
sporangiophore walls are consistent with the “acid growth” hypothesis [9]. An important difference is
that the duration of the creep for the sporangiophores’ walls is limited to less than ten minutes, compared
to hours for cucumber hypocotyls and oat coleoptiles. The finding that boiling the sporangiophore walls
for 15 s eliminates creep may suggest that protein activity mediates the creep response, but other
explanations must also be considered until more experiments are conducted.
The finding that the duration of the creep for the sporangiophores’ walls is limited to less than ten
minutes, compared to hours for cucumber hypocotyls and oat coleoptiles, is interesting and probably
significant. This finding, together with the fact that the molecular polymers, structure and endogenous
enzymes in the sporangiophore’s wall are different from those in plant and algal cell walls, may indicate
that the molecular wall loosening mechanism is different for fungal cells. It may be significant that the
duration of elongation growth during anoxia and the duration of creep in frozen-thawed walls sections
are both on the order of ten minutes. Interestingly, the duration of creep produced by decreasing the
pH is approximately the same as the duration of the light growth response and avoidance growth
response (transient increases in elongation growth rate) exhibited by the sporangiophores [7,17,18].
A mechanism is suggested by these findings in which proton effluxes mediate sensory growth responses of
the sporangiophores. One may speculate that the sporangiophore’s protoplast excretes protons at an
increased rate onto the inner surface of the growth zone wall to elicit a transient increase in irreversible
wall extensibility [27,28] that produces a transient increase in elongation growth rate, without increasing
the turgor pressure [29]; see Equation (4). The phototropic response (growing towards a light source)
and avoidance response (growing away from solid objects) could be produced by spatially increasing
the proton efflux on the distal side (phototropic response) and proximal side (avoidance response) of the
sporangiophore’s growth zone. Future research will include adding wall building substrates, activators
and endogenous wall enzymes to the bathing solution during constant-tension extension experiments to
learn if they will increase the duration of creep in the frozen-thawed sporangiophore wall.
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4. Experimental Section
4.1. Biological Material
The wild-type strain, NRRL1555(−), of P. blakesleeanus was obtained from the Biology Division,
California Institute of Technology, Pasadena, USA. Potato dextrose agar was prepared and sterilized in
12 × 35 mm glass vials. Vegetative spores were heat-shocked in a water bath held at approximately
45 °C for 20 min. After heat-shocking, the spores were triturated and inoculated on the potato dextrose
agar in the glass vials. The sporangiophore cultures in the vials were kept at 21 °C ±0.5 °C in a ventilated,
wood chamber and illuminated from above by four fluorescent lamps. Three days after inoculation,
sporangiophores begin to appear. Long sporangiophores are removed from the mycelium (plucked) daily
to obtain big and robust sporangiophores for experimentation on the next day. Typically sporangiophores
from 4 to 6 day-old cultures are used for experimentation.
4.2. Anoxia Experiments
4.2.1. Elongation Growth Measurements
The elongation growth is determined by measuring the change in length of the sporangiophore, ΔL,
at one-minute time intervals. The change in length is measured using a long focal length horizontal
microscope (Gaertner; 7011K eyepiece and 32 m/m EFL objective) mounted to a 3D micromanipulator
(Line Tool Co., Allentown, PA, USA; Model H-2, with digital micrometers). An electronic timer is used
to measure the time intervals.
4.2.2. Turgor Pressure Measurements
The turgor pressure of the sporangiophore is measured with a manual version of the pressure
probe [20]. A gage pressure transducer is used in the pressure probe, which measured the difference
between the absolute pressure and the local atmospheric pressure; the gage pressure transducer was
purchased from Kulite Semiconductor Products, Ridgefteld, NJ, USA (Model XT-190-300G), and
calibrated inside the pressure probe using a Heise Bourdon Tube Pressure Gauge (Dresser Industries,
Newton, CT, USA; Model CMM, 0-200 PSIG Range). The transducer’s output is recorded on a Houston
Omniscribe Stripchart Recorder (Ametek, Berwyn, PA, USA; Model D5217-2). The pressure probe was
mounted on a 3D micromanipulator so that the micro-capillary tip (typically 5–10 μm outer diameter)
can be guided to impale the sporangiophore under visual observation using a horizontally-mounted
EZM-2TR Trinocular Zoom Stereomicroscope (Meiji Labax Co., Tokyo, Japan). The micro-capillary of
the pressure probe was filled with inert silicone oil (Dow Corning Corp., Midland, MI, USA; fluid 200,
1–2 centistoke viscosity). After the cell was impaled, the cell sap-oil interface was maintained at a
fixed location within the micro-capillary tip to measure the turgor pressure of the sporangiophore.
Turgor pressure step-ups were produced by advancing a manually-controlled control rod within the
pressure probe to inject inert silicone oil into the vacuole.
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4.2.3. Experimental Protocol
A Stage IV sporangiophore was selected and placed in an experimental chamber that was constructed
for these experiments (Figure 8). The sporangiophore was adapted in the experimental chamber to
atmospheric air, constant and symmetric distribution of light and other environmental conditions for
20 min. The light and environmental conditions were maintained constant throughout the adaptation
period and during the experiment to prevent growth responses. After the adaptation, measurement of the
elongation growth was initiated and continued for the remainder of the experiment using a long focal
length horizontal microscope. After a period of time (usually 10 min), the sporangiophore stalk was
impaled with the pressure probe’s micro-capillary tip with the assistance of a horizontally-mounted
stereomicroscope, and the turgor pressure was continuously measured and monitored with the pressure
probe for the remainder of the experiment. Then, both the elongation rate and turgor pressure were
monitored for a period of time to ensure that the sporangiophore was growing at a normal rate.
Afterwards, nitrogen gas was injected from the bottom of the chamber to fill the apparatus chamber
containing the sporangiophore and to create an anoxic environment. The oxygen concentration was
measured and monitored with a meter (Model 50SD-2, 0–20 mL/min, McMillan Company, Georgetown,
TX, USA) inside the chamber and located next to the sporangiophore’s growth zone. Typically, the
oxygen concentration decreased from approximately 21% to less than 1% within a minute after the
nitrogen gas was injected into the chamber. Nitrogen gas was continuously injected into the chamber at
a low rate to maintain an oxygen concentration level of less than 1%.
4.3. Frozen Thawed Experiments
Sporangiophores, which are frozen for one or two days and then thawed, were used for these experiments.
A 5-mm section from the sporangiophore stalk that contains the growth zone was cut with a razor blade
and used for experimentation.
4.3.1. Experimental Apparatus
A small acrylic rectangular box was constructed and used as the testing apparatus for the extension
experiments (Figure 9). In the testing apparatus, the frozen-thawed wall section is attached to two glass
slides, one at each end (glued with Gorilla Super Glue). One glass slide is attached to the testing
apparatus and fixed. The other glass slide is attached to a small weight (1.24 g) with a string and is free
to slide within the box. The weight is supported on a platform to prevent tension on the wall section
before the extension protocol is initiated. A microscope assembly attached to a micrometer is used to
measure the change in length of the wall section (Figure 9A). A reference point (end of the wall section)
is used throughout the experiment to measure the change in length of the wall section as a function of
time. Initially, the frozen-thawed wall section is aligned to the reference point in the microscope, so that
changes in length can be measured during the experiment by realigning to the reference point every
minute and measuring the change on the digital micrometer.
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Figure 8. An illustration of the experimental chamber used for anoxia experiments. A vial
containing a sporangiophore is inserted into a holder inside the chamber. Afterwards, a back
support is advanced manually to the sporangiophore stalk. The back support keeps the stalk
stationary while the micro-capillary tip of the pressure probe is advanced to impale the
stalk below the growth zone. Two people are required to conduct a single experiment. One
person uses a stereomicroscope to impale the sporangiophore stalk, monitor the cell sap-oil
interface and generally operate the pressure probe. On the opposite side, another person uses
a horizontal microscope, attached to a 3D micromanipulator with digital micrometers, to
measure the change in elongation as a function of time.
4.3.2. Experimental Protocol
A Stage IV sporangiophore is selected and frozen for at least 24 h. A 5-mm wall section that includes
the growth zone of the frozen sporangiophore is removed with a razor blade. Then, each end of the wall
section is glued to a glass slide inside the testing apparatus, one of which is fixed (stationary) and the
other (attached to a weight by a string) is free to slide. The frozen wall section thaws while the glue dries.
After the glue is dry and the wall section is securely clamped to the two glass slides, distilled water is
added to the apparatus until the top part of the apparatus is half full and left to adapt for 10 min (Figure 9A).
Following this adaptation period, the change in length is measured every minute for the remainder of the
experiment, and the platform supporting the weight is slowly lowered to apply tension to the wall section
until the weight hangs freely. Five minutes after the wall section has been in constant tension, a
predetermined amount of buffer solution was added to the water to lower the pH to approximately 4.6. A
pH pen (Large Display ATC pH Pen, Model 850051, VWR International) is used to measure the pH of
the bathing solution when the buffer solution is first added and at the end of the experiment. The change
in length is measured continuously until no extension is observed (Figure 9B). The experimental protocol
for the frozen-thawed-boiled experiments is the same, except that after removing the sporangiophore
from the freezer, it is transferred into a water bath and boiled for 15 s using a microwave before the
extension protocol is conducted.
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Figure 9. Schematic illustration of the experimental set-up used for the constant-tension extension
experiments. (A) Testing arrangement for the frozen-thawed wall section during the
adaptation phase. The testing apparatus is filled with water, and the platform supporting the
weight is not lowered (right side). A microscope view of the aligning process (left side) of the
frozen-thawed wall section to the reference point in the microscope, so that changes in length
can be measured during the experiment by realigning to the reference point every minute
and measuring the change in length on the digital micrometer; (B) Testing arrangement for
the frozen-thawed wall section during the extension testing phase. The platform is lowered,
and the weight hangs freely during the test (right side). Buffer solution (pink) is added to the
testing apparatus to lower the pH to 4.6. A microscope view of the wall section in tension is
depicted (left side).
5. Conclusions
Experiments that used anoxia to terminate the metabolism of the protoplasm of Stage I and Stage IV
sporangiophores of P. blakesleeanus and chemically isolate their walls reveal a wall chemistry and
chemorheology that continues for ten minutes or longer to produce elongation and irreversible wall extension
(Figures 3–5 and Table 1). Constant-tension extension experiments were conducted on frozen-thawed
wall sections of Stage IV sporangiophores to investigate the wall chemistry and chemorheology revealed
in the anoxia experiments. The objective was to learn if lowering the pH of the bathing solution from
7.0 to 4.6 will elicit irreversible extension and creep. The results (Figures 6 and 7 and Table 2) demonstrate
that lowering the pH produces irreversible wall extension and creep in the frozen-thawed wall sections
(which include the growth zone) of the Stage IV sporangiophore. This finding is consistent with the
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“acid growth” hypothesis that was proposed for plant cell walls [9]. Interestingly, frozen-thawed wall
sections that were subject to boiling water for 15 s do not exhibit creep after the pH is lowered to 4.6.
This finding suggests that protein activity may be involved in the observed creep response, but other
experimental results are needed to support this suggestion. Except for the short duration of creep, the
constant tension-extension behavior of the frozen-thawed and frozen-thawed-boiled wall sections of
the Stage IV sporangiophore is qualitatively similar to that obtained from cucumber (Cucumis sativus L.)
hypocotyls and oat (Avena sativa L.) coleoptiles [10,11]. It is thought that the limited and short duration
of creep exhibited by the frozen-thawed sporangiophore wall to acidic pH may be significant. This
finding, together with the fact that the molecular polymers, structure and endogenous enzymes in the
sporangiophore walls are different from those in plant and algal cell walls, may suggest that the
molecular wall loosening mechanism is different from those of higher plant cells and algal cells.
Acknowledgments
This research was supported by an NSF Grant, MCB-0948921, awarded to Joseph K.E. Ortega.
Cindy M. Munoz acknowledges the support from an NSF Graduate STEM fellowship in K-12 Grant,
DGE-0742434. Cindy M. Munoz and David G. Ramirez acknowledge the support from an NSF Bridge
to the Doctorate Grant, HRD-1301885 (sub-award, G-8960-1). The authors acknowledge the early
research conducted by Jessica E.C. Olson, Elena L. Ortega and Morgan A. Scott, who studied the
elongation and turgor pressure behavior of the sporangiophores of P. blakesleeanus during anoxia.
Furthermore, the authors acknowledge that Jessica Olson and Elena Ortega conducted the experiment
whose results are shown in Figure 2 and that Morgan Scott and Elena Ortega conducted early turgor
pressure step-up experiments whose results are similar to those shown in Figure 4, but not the results
used in Figure 4. Other interesting results are reported in a master thesis by Jessica Olson and a master
report by Morgan Scott. Olson, Jessica E.C. Phycomyces: Helical growth and turgor pressure behavior
during death by asphyxiation. M.S. thesis, University of Colorado, Denver, 2000, 1–164. Scott, Morgan
A. Phycomyces blakesleeanus: An investigation of elongation growth and turgor pressure of wild-type
sporangiophores during anoxia, and the growth and physiological characteristics of two mutant
sporangiophores. M.E. Report, University of Colorado Denver, 2008, 1–77.
Author Contributions
Joseph K.E. Ortega initiated, supervised and provided financial support for the all of the research
conducted and reported in this paper. In addition, he was predominately responsible for the development
of the experimental protocols, directing the analysis of the data, interpretation of the results and writing
the paper. Jason T. Truong, Cindy M. Munoz and David G. Ramirez conducted all of the experiments
whose results are reported in this paper (except for Figure 2) and contributed equally to this work.
In addition, these authors conducted most of the data analysis, assisted in the development of the
experimental protocols and assisted in the writing of the text in the Materials and Methods.
Conflicts of Interest
The authors declare no conflict of interest.
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